Skip to main content
NCBI home page
The EMBO Journal logoLink to The EMBO Journal
. 1990 Sep;9(9):2679–2684. doi: 10.1002/j.1460-2075.1990.tb07453.x

Mutational analysis of ligand binding activity of beta 2 adrenergic receptor expressed in Escherichia coli.

R M Breyer 1, A D Strosberg 1, J G Guillet 1
PMCID: PMC551972  PMID: 2167830

Abstract

A novel in situ screening procedure was used to identify neutral mutations in the human beta 2 adrenergic receptor (beta 2AR). The coding region of the human beta 2AR gene was subcloned under transcriptional control of an inducible T7 promoter and used to transform Escherichia coli. Colonies expressing the beta 2AR bound the radiolabeled antagonist [125I]iodocyanopindolol and could be identified by autoradiography after transfer to nitrocellulose filters. The region of the beta 2AR between residues 76 and 83, in the second transmembrane helix, was mutagenized by a saturation mutagenesis technique, so that virtually all of the beta 2AR genes contained at least one mutation. Colonies retaining ligand binding activity were isolated using the in situ screen. Sequence analysis of the active mutant receptor genes allowed the identification of individual amino acid side chains which are essential for either ligand binding or structural integrity of the beta 2AR receptor.

Full text

PDF
2679

Images in this article

2679Fig. 1.
on p.2679
2680Fig. 2.
on p.2680
2681Fig. 3.
on p.2681

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Amann E., Brosius J., Ptashne M. Vectors bearing a hybrid trp-lac promoter useful for regulated expression of cloned genes in Escherichia coli. Gene. 1983 Nov;25(2-3):167–178. doi: 10.1016/0378-1119(83)90222-6. [DOI] [PubMed] [Google Scholar]
  2. Bouvier M., Hnatowich M., Collins S., Kobilka B. K., Deblasi A., Lefkowitz R. J., Caron M. G. Expression of a human cDNA encoding the beta 2-adrenergic receptor in Chinese hamster fibroblasts (CHW): functionality and regulation of the expressed receptors. Mol Pharmacol. 1988 Feb;33(2):133–139. [PubMed] [Google Scholar]
  3. Bowie J. U., Sauer R. T. Identifying determinants of folding and activity for a protein of unknown structure. Proc Natl Acad Sci U S A. 1989 Apr;86(7):2152–2156. doi: 10.1073/pnas.86.7.2152. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Breyer R. M., Sauer R. T. Mutational analysis of the fine specificity of binding of monoclonal antibody 51F to lambda repressor. J Biol Chem. 1989 Aug 5;264(22):13355–13360. [PubMed] [Google Scholar]
  5. Chapot M. P., Eshdat Y., Marullo S., Guillet J. G., Charbit A., Strosberg A. D., Delavier-Klutchko C. Localization and characterization of three different beta-adrenergic receptors expressed in Escherichia coli. Eur J Biochem. 1990 Jan 12;187(1):137–144. doi: 10.1111/j.1432-1033.1990.tb15287.x. [DOI] [PubMed] [Google Scholar]
  6. Chung F. Z., Wang C. D., Potter P. C., Venter J. C., Fraser C. M. Site-directed mutagenesis and continuous expression of human beta-adrenergic receptors. Identification of a conserved aspartate residue involved in agonist binding and receptor activation. J Biol Chem. 1988 Mar 25;263(9):4052–4055. [PubMed] [Google Scholar]
  7. Dixon R. A., Kobilka B. K., Strader D. J., Benovic J. L., Dohlman H. G., Frielle T., Bolanowski M. A., Bennett C. D., Rands E., Diehl R. E. Cloning of the gene and cDNA for mammalian beta-adrenergic receptor and homology with rhodopsin. Nature. 1986 May 1;321(6065):75–79. doi: 10.1038/321075a0. [DOI] [PubMed] [Google Scholar]
  8. Dixon R. A., Sigal I. S., Candelore M. R., Register R. B., Scattergood W., Rands E., Strader C. D. Structural features required for ligand binding to the beta-adrenergic receptor. EMBO J. 1987 Nov;6(11):3269–3275. doi: 10.1002/j.1460-2075.1987.tb02645.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Dixon R. A., Sigal I. S., Rands E., Register R. B., Candelore M. R., Blake A. D., Strader C. D. Ligand binding to the beta-adrenergic receptor involves its rhodopsin-like core. Nature. 1987 Mar 5;326(6108):73–77. doi: 10.1038/326073a0. [DOI] [PubMed] [Google Scholar]
  10. Dohlman H. G., Bouvier M., Benovic J. L., Caron M. G., Lefkowitz R. J. The multiple membrane spanning topography of the beta 2-adrenergic receptor. Localization of the sites of binding, glycosylation, and regulatory phosphorylation by limited proteolysis. J Biol Chem. 1987 Oct 15;262(29):14282–14288. [PubMed] [Google Scholar]
  11. Dohlman H. G., Caron M. G., Lefkowitz R. J. A family of receptors coupled to guanine nucleotide regulatory proteins. Biochemistry. 1987 May 19;26(10):2657–2664. doi: 10.1021/bi00384a001. [DOI] [PubMed] [Google Scholar]
  12. Emorine L. J., Marullo S., Delavier-Klutchko C., Kaveri S. V., Durieu-Trautmann O., Strosberg A. D. Structure of the gene for human beta 2-adrenergic receptor: expression and promoter characterization. Proc Natl Acad Sci U S A. 1987 Oct;84(20):6995–6999. doi: 10.1073/pnas.84.20.6995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Fraser C. M., Chung F. Z., Wang C. D., Venter J. C. Site-directed mutagenesis of human beta-adrenergic receptors: substitution of aspartic acid-130 by asparagine produces a receptor with high-affinity agonist binding that is uncoupled from adenylate cyclase. Proc Natl Acad Sci U S A. 1988 Aug;85(15):5478–5482. doi: 10.1073/pnas.85.15.5478. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Fraser C. M. Site-directed mutagenesis of beta-adrenergic receptors. Identification of conserved cysteine residues that independently affect ligand binding and receptor activation. J Biol Chem. 1989 Jun 5;264(16):9266–9270. [PubMed] [Google Scholar]
  15. Frielle T., Daniel K. W., Caron M. G., Lefkowitz R. J. Structural basis of beta-adrenergic receptor subtype specificity studied with chimeric beta 1/beta 2-adrenergic receptors. Proc Natl Acad Sci U S A. 1988 Dec;85(24):9494–9498. doi: 10.1073/pnas.85.24.9494. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Kobilka B. K., Dixon R. A., Frielle T., Dohlman H. G., Bolanowski M. A., Sigal I. S., Yang-Feng T. L., Francke U., Caron M. G., Lefkowitz R. J. cDNA for the human beta 2-adrenergic receptor: a protein with multiple membrane-spanning domains and encoded by a gene whose chromosomal location is shared with that of the receptor for platelet-derived growth factor. Proc Natl Acad Sci U S A. 1987 Jan;84(1):46–50. doi: 10.1073/pnas.84.1.46. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kobilka B. K., Kobilka T. S., Daniel K., Regan J. W., Caron M. G., Lefkowitz R. J. Chimeric alpha 2-,beta 2-adrenergic receptors: delineation of domains involved in effector coupling and ligand binding specificity. Science. 1988 Jun 3;240(4857):1310–1316. doi: 10.1126/science.2836950. [DOI] [PubMed] [Google Scholar]
  18. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  19. Loeb D. D., Swanstrom R., Everitt L., Manchester M., Stamper S. E., Hutchison C. A., 3rd Complete mutagenesis of the HIV-1 protease. Nature. 1989 Aug 3;340(6232):397–400. doi: 10.1038/340397a0. [DOI] [PubMed] [Google Scholar]
  20. Marullo S., Delavier-Klutchko C., Eshdat Y., Strosberg A. D., Emorine L. Human beta 2-adrenergic receptors expressed in Escherichia coli membranes retain their pharmacological properties. Proc Natl Acad Sci U S A. 1988 Oct;85(20):7551–7555. doi: 10.1073/pnas.85.20.7551. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Marullo S., Emorine L. J., Strosberg A. D., Delavier-Klutchko C. Selective binding of ligands to beta 1, beta 2 or chimeric beta 1/beta 2-adrenergic receptors involves multiple subsites. EMBO J. 1990 May;9(5):1471–1476. doi: 10.1002/j.1460-2075.1990.tb08264.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Oliphant A. R., Nussbaum A. L., Struhl K. Cloning of random-sequence oligodeoxynucleotides. Gene. 1986;44(2-3):177–183. doi: 10.1016/0378-1119(86)90180-0. [DOI] [PubMed] [Google Scholar]
  23. Oliphant A. R., Struhl K. An efficient method for generating proteins with altered enzymatic properties: application to beta-lactamase. Proc Natl Acad Sci U S A. 1989 Dec;86(23):9094–9098. doi: 10.1073/pnas.86.23.9094. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Rees D. C., DeAntonio L., Eisenberg D. Hydrophobic organization of membrane proteins. Science. 1989 Aug 4;245(4917):510–513. doi: 10.1126/science.2667138. [DOI] [PubMed] [Google Scholar]
  25. Reidhaar-Olson J. F., Sauer R. T. Combinatorial cassette mutagenesis as a probe of the informational content of protein sequences. Science. 1988 Jul 1;241(4861):53–57. doi: 10.1126/science.3388019. [DOI] [PubMed] [Google Scholar]
  26. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Strader C. D., Dixon R. A., Cheung A. H., Candelore M. R., Blake A. D., Sigal I. S. Mutations that uncouple the beta-adrenergic receptor from Gs and increase agonist affinity. J Biol Chem. 1987 Dec 5;262(34):16439–16443. [PubMed] [Google Scholar]
  28. Strader C. D., Sigal I. S., Candelore M. R., Rands E., Hill W. S., Dixon R. A. Conserved aspartic acid residues 79 and 113 of the beta-adrenergic receptor have different roles in receptor function. J Biol Chem. 1988 Jul 25;263(21):10267–10271. [PubMed] [Google Scholar]
  29. Strader C. D., Sigal I. S., Register R. B., Candelore M. R., Rands E., Dixon R. A. Identification of residues required for ligand binding to the beta-adrenergic receptor. Proc Natl Acad Sci U S A. 1987 Jul;84(13):4384–4388. doi: 10.1073/pnas.84.13.4384. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Studier F. W., Moffatt B. A. Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J Mol Biol. 1986 May 5;189(1):113–130. doi: 10.1016/0022-2836(86)90385-2. [DOI] [PubMed] [Google Scholar]
  31. Zagursky R. J., Berman M. L. Cloning vectors that yield high levels of single-stranded DNA for rapid DNA sequencing. Gene. 1984 Feb;27(2):183–191. doi: 10.1016/0378-1119(84)90139-2. [DOI] [PubMed] [Google Scholar]

Articles from The EMBO Journal are provided here courtesy of Nature Publishing Group

RESOURCES